Platelet transfusion is only one therapy against thrombocytopenia caused by e.g., bleeding associated with accidents and during use of anti-cancer agents. Platelet preparations to be used for the time are produced completely (100%) depending upon blood donation with good intentions, at present. Platelets are very fragile and a method enabling platelets for use in therapy to store for a long time has not yet been developed. Actually, it is reported that the storage life of platelets in the latest medical institutions is four days; however, in consideration of time required for inspection and shipment, substantial storage life thereof in clinical sites including clinics is conceivably about three days. Likewise, many blood banks have a difficulty in storing platelets while keeping freshness at all times. In addition, the supply amount of platelet preparations is likely to vary dependent upon a decrease of blood donors and an increase of blood donors affected with viral infectious diseases.
In the circumstances, recently, a novel platelet supply source has attracted attention, which has been developed in place of blood donation having such problems (non-patent document 1). As an example, development of a technique of producing a large amount of platelets in vitro using somatic stem cells, i.e., hematopoietic stem cells (umbilical cord blood stem cells) is known. However, this technique has not yet been put into practical use, because an in vitro method for proliferating hematopoietic stem cells per se has not yet been established. In contrast, pluripotent stem cells, i.e., embryonic stem (ES) cells have an advantage in that they can be unlimitedly proliferated in vitro and have attracted attention as a supply source for producing blood cells including platelets. In this respect, techniques for producing mature megakaryocytes and platelets from human ES cells have been already reported (non-patent document 2). However, in the techniques (methods), the production efficiency of platelets is low and tens of thousands of petri dishes are required for producing a single blood transfusion preparation. These methods were insufficient from a practical point of view.
In transfusion of platelets, refractory to platelet transfusion is raised as a problem. At the first-time transfusion, platelets having a different human leukocyte antigen (HLA) from a patient can be used; however, a specific antibody against the HLA is produced in the patient's body when transfusion is repeated, with the result that the platelets are rejected immediately upon transfusion. In addition, since platelets have own blood type, i.e., an allogeneic human platelet antigen (HPA), refractory to transfusion caused by incompatibility of HPA types is also known. As a technique which can overcome this problem, techniques for producing megakaryocytes and platelets from human induced pluripotent stem (iPS) cells have been reported (non-patent document 3). For example, if platelets are induced from a patient-derived iPS cells, it is theoretically possible to produce a rejection-free custom-made platelet preparation. However, in producing platelets from iPS cells, at least about 50 days are required for producing platelets from fibroblasts (non-patent document 3). For the reason, this production method was insufficient from a practical point of view. In the meantime, as a method for producing platelets from fibroblasts, a technique called direct reprogramming is known (non-patent document 4). According to this technique, the period required for producing platelets will be greatly shorter than the method for producing platelets via iPS cells. Advantageously, platelets are produced in about 14 days. However, the direct reprogramming using fibroblasts requires gene introduction. The effect of the presence of a gene transfer vector on safety is concerned.
As a culture solution for inducing differentiation of hematopoietic stem cells into megakaryocytes, platelets, MKLI medium (megakaryocyte lineage induction medium) is known. The MKLI medium is a medium prepared by adding, 2 mM L-glutamine, a 100 U/mL penicillin-streptomycin solution, 0.5% bovine serum albumin, 4 μg/mL LDL cholesterol, 200 μg/mL iron-saturated transferrin (iron-bound transferrin), 10 μg/mL insulin, 50 μM 2-β-mercaptoethanol, nucleotides (20 μM for each of ATP, UTP, GTP and CTP) and 50 ng/mL thrombopoietin (thrombopoietin: TPO) to Iscove's Modified Dulbecco's Medium (IMDM) (non-patent document 5). The present inventors have so far conducted studies on a technique for inducing differentiation of cells excluding hematopoietic stem cells into megakaryocytes, platelets. As a result, they have found that if preadipocytes derived from a human subcutaneous adipose tissue (non-patent documents 5, 6) and mouse-derived preadipocytes (non-patent documents 5, 7) are cultured in the MKLI medium, they can be differentiated into megakaryocytes, platelets. The present inventors further conducted studies and have found a further excellent method for successfully producing megakaryocytes and/or platelets (Patent Document 1). The production method of Patent Document 1 is a production method comprising culturing mesenchymal cells in a mesenchymal cell culturing basic medium comprising an iron ion and an iron transporter, and collecting megakaryocytes and/or platelets from a culture. In the production method of Patent Document 1, it is possible to produce megakaryocytes having the ability to make platelets and/or platelets having thrombus forming ability from mesenchymal cells such as preadipocytes in a relatively short period of time, simply and in a large amount as well as at lower cost or more efficiently in vitro even if e.g., TPO is not added to the medium.
As described above, the production method of Patent Document 1 is an excellent production method, which overcame drawbacks of conventional production methods for platelets using hematopoietic stem cells, ES cells or iPS cells. However, as the results of analysis (made by the present inventors) for the relationship between the type of surface antigen of mesenchymal cells, such as preadipocytes (before differentiation induction into megakaryocytes or platelets) and the differentiation efficiency of the mesenchymal cells into e.g., megakaryocytes in differentiation induction into e.g., megakaryocytes, it was shown that c-MPL receptor-positive mesenchymal cells are differentiated into megakaryocytes or platelets with extremely high efficiency, compared to c-MPL receptor-negative mesenchymal cells. However, the ratio of c-MPL receptor positive cells in mesenchymal cells such as preadipocytes was extremely low: about 0.5 to 1%.
A c-MPL receptor is known as an in vivo TPO receptor. More specifically, TPO is known as a ligand of a c-MPL receptor in vivo. c-MPL is a glycoprotein, which expresses on hematopoietic stem cells and megakaryocyte lineage cells and belongs to a cytokine receptor gene family. It has been suggested c-MPL is deeply involved in platelet production as a receptor for a novel factor involved in platelet production. TPO is synthesized in the liver as a proprotein consisting of 353 amino acids and becomes a mature protein molecule by cleaving a signal peptide of 21 amino acids. The mature protein molecule consists of two domains having a high homology with erythropoietin and a highly glycosylated carboxy terminal important for protein stability (non-patent document 8). It is reported that, in a TPO gene or c-MPL receptor gene-defective mouse, the number of platelets decreases down to about 10 to 20% of that of a wild type mouse (Non-Patent Document 9). From this, it is suggested that TPO and a c-MPL receptor are involved in regulation of the pathway for producing platelets from hematopoietic stem cells.
In Non-Patent Document 10, it is described that the activity of the promotor of a c-MPL receptor gene in human megakaryoblastic leukemia cell line, i e., CMK cells, is induced by TPO. In Non-Patent Document 10, the activity of the promotor of a c-MPL receptor gene is measured based on the luciferase activity by the expression of a luciferase gene ligated to a portion downstream of the promoter. However, in Non-Patent Document 10, it is not to say that the effect of TPO on expression of a c-MPL receptor on a cell surface is confirmed. In contrast, Non-Patent Document 11 shows that TPO reduces expression of a c-MPL receptor on the surface of CMK cells.